1. Field of the Invention
The present invention relates to vaccines using viral antigens, and in particular, to vaccines for the treatment and prevention of human immunodeficiency virus (HIV) infection. The vaccines of this invention comprise alphavirus RNA replicon systems which contain nucleic acid sequence encoding antigens for eliciting an immune response to HIV.
2. Background
The successful control of the AIDS epidemic will require an effective vaccine for human immunodeficiency virus type 1 (HIV) that significantly reduces or prevents the spread of infection. Currently, several viral vector systems as well as naked DNA are at various stages of pre-clinical and clinical evaluation as candidate HIV vaccines. Recombinant poxviruses are the most widely studied virus vectors and are furthest along in clinical development (e.g., ALVAC).
The alphavirus-based replicon particle systems, such as the ones described in U.S. Pat. No. 5,792,462 and herein referred to as xe2x80x9cVRPs,xe2x80x9d have multiple distinct properties that make them attractive as an HIV vaccine delivery technology. These properties include: natural targeting to and expression in lymphoid tissues (an optimal site for induction of an immune response); high antigen expression levels, e.g., up to 20% of total cell protein; induction of balanced humoral, cellular, and mucosal immune responses; sustained efficacy over multiple simultaneous or sequential inoculations of the vector; and a high margin of safety.
Venezuelan equine encephalitis virus (VEE) is a member of the Alphaviruses group, which also includes the prototype Sindbis virus (SIN) and Semliki Forest virus (SFV), and is comprised of enveloped viruses containing plus-stranded RNA genomes within icosahedral capsids (Strauss, 1994). Alphavirus genomes are: approximately 11.5 kb long, capped, polyadenylated, and infectious under appropriate transfection conditions. The nucleocapsid is composed of 240 molecules of the capsid protein arranged as a T=4 icosahedron, and is surrounded by a lipoprotein envelope (Paredes et al., 1993). Protruding from the virion surface are 80 glycoprotein spikes, each of which is a trimer of virally encoded E1 and E2 glycoprotein heterodimers. The virions contain no host proteins.
Alphaviruses share replication strategies and genomic organization. The complete replicative cycle of alphaviruses occurs in the cytoplasm of infected cells. Expression from the alphavirus genome is segregated into two regions. The four enzymatic nonstructural proteins (nsP1-nsP4) are synthesized from the 5xe2x80x2 two-thirds of the genome-length RNA and are required for RNA replication. Immediately following infection, the nsPs are produced by translation of parental genomes and catalyze the synthesis of a full-length negative-sense copy of the genome. This serves as a template for the synthesis of progeny plus-stranded genomes.
The negative-sense copy of the genome also serves as the template for the synthesis of subgenomic mRNA at approximately 10-fold molar excess relative to genomic RNA in infected cells (Schlesinger and Schlesinger, 1990). Synthesis of subgenomic 26S mRNA is initiated from the highly active internal 26S mRNA promoter, which is functional only on the negative-sense RNA. The subgenomic mRNA corresponds to the 3xe2x80x2 one-third of the genome and encodes the alphavirus structural proteins.
Full-length, infectious cDNA clones of the RNA genome of VEE Davis et al., 1989) have been constructed, a panel of mutations which strongly attenuate the virus have been identified (Johnston and Smith, 1988; Davis et al., 1990), and various constellations of these attenuating mutations have been inserted into the clones to generate several live attenuated VEE vaccine candidates (Davis et al., 1991; 1995b; Grieder et al., 1995). The resulting vaccine candidates are avirulent and provide complete protection against lethal virus challenge in rodents, horses and nonhuman primates.
The alphavirus VRPs are propagation defective, single cycle vectors that contain a self-amplifying alphavirus RNA (replicon RNA) in which the structural protein genes of the virus are replaced by a heterologous antigen gene to be expressed. Alphavirus VRPs are typically made in cultured cells, referred to as packaging cells. Following introduction into mammalian cells, the replicon RNA is packaged into VRP by supplying the structural proteins in xe2x80x9ctrans,xe2x80x9d i.e., the cells are co-transfected with both replicon RNA and one or more separate helper RNAs which together encode the full complement of alphavirus structural proteins. Importantly, only the replicon RNA is packaged into VRP, as the helper RNA(s) lack the cis-acting packaging sequence required for encapsidation. Thus, the VRPs are defective, in that they can only infect target cells in culture or in vivo, where they express the heterologous antigen gene to high level, but they lack critical portions of the VEE genome (i.e., the VEE structural protein genes) necessary to produce virus particles which could spread to other cells.
Delivery of the replicon RNA into target cells (for vaccination) is facilitated by the VRP following infection of the target cells. In the cytoplasm of the target cell, the replicon RNA is first translated to produce the viral replicase proteins necessary to initiate self-amplification and expression. The heterologous antigen gene is encoded by a subgenomic mRNA, abundantly transcribed from the replicon RNA, leading to high level expression of the heterologous antigen gene product. Since the VEE structural protein genes are not encoded by the replicon RNA delivered to the target cell, progeny virion particles are not assembled, thus limiting the replication to a single cycle within the infected target cell. Experimental VRP vaccines have been successful in vaccinating rodents against influenza virus, Lassa fever virus and Marburg virus (Pushko et al., 1997; Hevey et al., 1998). In nonhuman primates, VRP vaccines have demonstrated complete efficacy against lethal Marburg virus challenge (Hevey et al., 1998), shown partial but significant protection against SIV infection and disease (Davis et al., 2000) and induced an anti-HA response at a level consistent with protection of humans against influenza virus infection.
The alphavirus based replicon vector systems, and in particular the VEE-based systems, present several advantages in vaccination, including safety and high immunogenicity/efficacy. VEE is unique among the alphaviruses in that a live attenuated IND VEE vaccine, TC-83, (Kinney et al., 1989; Kinney et al., 1993) has been inoculated into approximately 8,000 humans. This allows direct safety and efficacy comparisons between human, nonhuman primate and rodent responses to the same VEE derivative. A large body of experience strongly suggests that the animal models generally reflect the human susceptibility and disease course, except that mice are far more susceptible to lethal VEE disease than humans or nonhuman primates. Furthermore, the VEE replicon vectors express high levels of the gene of interest in cell culture, and in vivo expression is targeted to lymphoid tissues, reflecting the natural tropism mediated by the VEE glycoproteins. Cells in the draining lymph node of VRP-inoculated mice contain detectable amounts of the desired gene product within hours of inoculation. This expression continues for up to five days.
To date, VRP vector vaccines have been used in over 2000 rodents and in 94 macaques at doses up to 5xc3x97108 i.u., with no indication of any clinical manifestations.
In work reported by Pushko et al. (1997), individual mice were immunized sequentially with Lassa virus N-VRP and influenza virus HA-VRP. Groups of mice, which received two inoculations of 3xc3x97104 or 3xc3x97106 i.u. of Lassa N-VRP followed by two inoculations of 2xc3x97105 i.u. of HA-VRP, all responded with serum antibodies to both antigens. The level of anti-influenza antibody induced in these sequentially inoculated mice was equivalent to a control group, which received two inoculations of buffer followed by two inoculations of 2xc3x97105 i.u. of HA-VRP. All HA-VRP immunized mice were completely protected against influenza virus challenge. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Primary and booster immunization with a VRP preparation expressing an immunogen from one pathogen did not interfere with the development of a protective response to subsequent primary immunization and boosting with VRP expressing an immunogen from a second pathogen, thus showing that the VRP-based system can be used to induce immunity to a variety of pathogens in the same individual over time.
Four macaques were inoculated subcutaneously at week 0 with 105 i.u. each of SIV-gp160-VRP (env) and SIV MA/CA-VRP (gag), boosted by the same route at week 7 with 107 i.u. of each VRP vaccine, and intravenously at weeks 12 and 20 with 5xc3x97108 i.u. of each VRP. Two control animals were inoculated with equivalent doses of HA-VRP (haemagglutinin, a glycoprotein from influenza virus), and two with the vehicle only. The four SIV-VRPs immunized monkeys received subcutaneously an additional dose of 2xc3x97107 i.u. of gp140-VRP at week 41, followed by a final boost of 2xc3x97107 i.u. each of gp140-VRP and MA/CA-VRP at week 49. Four weeks after the final immunization, all eight macaques were challenged intravenously with the pathogenic virus, SIVsmE660.
After these inoculations, three of four test macaques had measurable CTL-specific killing directed against both SIV gag and env, all four had gp160 IgG antibody by ELISA, and the three animals which harbored SIV-specific CTL also showed neutralizing antibody to SIVsmH-4.
Four of four vaccinated animals were protected against disease for at least 16 months following intravenous challenge with the pathogenic SIV swarm, while the two vehicle controls required euthanasia at week 10 and week 11, post challenge. In two of the vaccinees, plasma virus levels were below the limit of detection by branched chain DNA assay. At 64 weeks post challenge, all four vaccinated animals showed no clinical signs of disease. One animal remained vDNA negative at 64 weeks.
The results of this highly pathogenic challenge demonstrated that the immune response induced by vaccination with SIV-VRP was effective in preventing early mortality and increasing the ability to suppress challenge virus replication. The ability to control SIV replication and reduce viral load to undetectable levels was closely correlated with the strongest measurable antibody and cellular immune responses.
While these results are encouraging, the level of protection obtained would not be acceptable for a human vaccine against HIV infection. Thus, there remains a need for a robust, effective and safe vaccine against HIV infection in humans. Development of a HIV vaccine comprising the complete, or immunogenic fragments of the, gag gene (Gag-VRP), an immunogenic portion of the pol gene (Pol-VRP), and the complete, or immunogenic fragments of the, env gene (Env-VRP), would increase the diversity of available CTL epitopes substantially and thus address this need.
The present invention provides a composition comprising two or more isolated nucleic acids selected from the group consisting of an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles by the gag gene product or the immunogenic fragment thereof and their release from a cell, and an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit reverse transcriptase activity.
Also provided is a composition comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles by the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to remove protease, integrase and RNase H regions and to inhibit reverse transcriptase activity, and wherein the nucleic acids are each contained within a separate alphavirus replicon particle.
In addition, the present invention provides a composition comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit reverse transcriptase activity, and wherein the nucleic acids are each contained within a separate alphavirus replicon particle, and further wherein the alphavirus replicon particles comprise a replicon RNA or at least one structural protein which comprises one or more attenuating mutations.
10. A method of making a population of alphavirus replicon particles of this invention is provided herein, comprising;
A) (a) providing a first helper cell for producing a first population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the first population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cells;
B) (a) providing a second helper cell for producing a second population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the second population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cells;
C) (a) providing a third helper cell for producing a third population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit reverse transcriptase activity, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the third population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cells; and
D) combining the first population of alphavirus particles produced from the first helper cell, the second population of alphavirus particles produced from the second helper cell and the third population of alphavirus particles produced from the third helper cell, thereby producing the population of alphavirus replicon particles.
Also provided is a method of making a population of alphavirus replicon particles, comprising:
A) (a) providing a first helper cell for producing a first population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the first population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cells;
B) (a) providing a second helper cell for producing a second population of infectious, replication defective alphavirus particle, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(g) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the second population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cells;
C) (a) providing a third helper cell for producing a third population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof is modified to inhibit reverse transcriptase activity, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the third population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cells; and
D) combining the first population of alphavirus particles produced from the first helper cell, the second population of alphavirus particles produced from the second helper cell and the third population of alphavirus particles produced from the third helper cell, thereby producing the population of alphavirus replicon particles.
Furthermore, the present invention provides a composition comprising two or more isolated nucleic acids selected from the group consisting of an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof.
In addition, the present invention provides a composition comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, and wherein the nucleic acids are each contained within a separate alphavirus replicon particle.
Also provided herein is a composition comprising a population of alphavirus replicon particles comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, and wherein the nucleic acids are each contained within a separate alphavirus replicon particle, and further wherein the alphavirus replicon particles comprise a replicon RNA or at least one structural protein which comprises one or more attenuating mutations.
In these embodiments, the gag gene product or immunogenic fragment thereof can be modified by mutation of the second codon, whereby a glycine is changed to an alanine and the pol gene product or immunogenic fragment thereof can be modified by mutation of the nucleotide sequence encoding the active site motif, whereby YMDD is changed to YMAA or HMAA. In addition, the pol gene product or immunogenic fragment thereof is modified to remove protease, integrase and RNase H regions and to produce only p51 of the pol gene product or immunogenic fragment thereof.
The present invention provides a method of making a population of alphavirus replicon particles, comprising:
A) (a) providing a first helper cell for producing a first population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the first population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cells;
B) (a) providing a second helper cell for producing a second population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the second population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cells;
C) (a) providing a third helper cell for producing a third population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the third population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cells; and
D) combining the first population of alphavirus particles produced from the first helper cell, the second population of alphavirus particles produced from the second helper cell and the third population of alphavirus particles produced from the third helper cell, thereby producing the population of alphavirus replicon particles.
An additional method of making a population of alphavirus replicon particles is provided, comprising:
A) (a) providing a first helper cell for producing a first population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the first population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cells;
B) (a) providing a second helper cell for producing a second population of infectious, replication defective alphavirus particle, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA,
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the second population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cells;
C) (a) providing a third helper cell for producing a third population of infectious, replication defective alphavirus particles, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the third population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture, and further wherein at least one of said replicon RNA, said first helper RNA, and said one or more additional helper RNA(s) comprises one or more attenuating mutations;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cells; and
D) combining the first population of alphavirus particles produced from the first helper cell, the second population of alphavirus particles produced from the second helper cell and the third population of alphavirus particles produced from the third helper cell, thereby producing the population of alphavirus replicon particles.
In each of the methods above, the alphavirus replicon RNA of at least one of the first helper cell, the second helper cell and the third helper cell can comprise sequence encoding at least one alphavirus structural protein and the first helper RNA and the one or more additional helper RNA(s) in the at least one of the first helper cell, the second helper cell and the third helper cell, can encode at least one other alphavirus structural protein not encoded by the replicon RNA.
Furthermore, in the methods above which recite attenuating mutations, only at least one of the first population of alphavirus particles, the second population of alphavirus particles and the third population of alphavirus particles can comprise particles wherein at least one of the replicon RNA, the first helper RNA, and the one or more additional helper RNA(s) comprises one or more attenuating mutations.
The present invention further provides alphavirus particles produced by any of the methods of this invention.
The present invention further provides a method of inducing an immune response to human immunodeficiency virus in a subject, comprising administering to the subject an immunogenic amount of the populations and/or compositions of this invention, in a pharmaceutically acceptable carrier.
Also provided herein is a method of treating or preventing infection by human immunodeficiency virus in a subject, comprising administering to the subject an immunogenic amount of the populations and/or compositions of this invention, in a pharmaceutically acceptable carrier.
Also provided by the present invention is an alphavirus replicon virosome comprising an alphavirus replicon RNA encapsidated by a lipid bilayer comprising alphavirus glycoproteins, E1 and E2, which in one embodiment, can be Venezuelan Equine Encephalitis glycoproteins E1 and E2.
A method of producing an alphavirus replicon virosome is further provided, comprising: a) combining alphavirus replicon RNA, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and b) gradually removing detergent, whereby alphavirus replicon virosomes are produced. Also provided is a virosome produced by this method.
Furthermore, the present invention provides a method of eliciting an immune response in a subject, comprising administering to the subject an immunogenic amount of the alphavirus replicon virosome of this invention in a pharmaceutically acceptable carrier.
The present invention additionally provides a method of treating or preventing infection by human immunodeficiency virus in a subject, comprising administering to the subject an immunogenic amount of the alphavirus replicon virosome of this invention, wherein the virosome comprises alphavirus replicon RNA encoding one or more HIV immunogens.
In further embodiments, the present invention provides a composition comprising a population of alphavirus replicon virosomes comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, and wherein the nucleic acids are each contained within a separate alphavirus replicon virosome.
Additionally provided herein is a composition comprising a population of alphavirus replicon virosomes comprising two or more isolated nucleic acids selected from the group consisting of 1) an isolated nucleic acid encoding an env gene product or an immunogenic fragment thereof of a human immunodeficiency virus, 2) an isolated nucleic acid encoding a gag gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, and 3) an isolated nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in inactivation of reverse transcriptase activity in the pol gene product or immunogenic fragment thereof, and wherein the nucleic acids are each contained within a separate alphavirus replicon virosome.
A method of producing a population of alphavirus replicon virosomes is provided herein, comprising:
A) (a) producing a first population of alphavirus replicon virosomes by combining alphavirus replicon RNA comprising nucleic acid encoding an env gene product or immunogenic fragment thereof, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and
b) gradually removing detergent, whereby alphavirus replicon virosomes are produced;
B) (a) producing a second population of alphavirus replicon virosomes by combining alphavirus replicon RNA comprising nucleic acid encoding a gag gene product or immunogenic fragment thereof, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and
b) gradually removing detergent, whereby alphavirus replicon virosomes are produced;
C) (a) producing a third population of alphavirus replicon virosomes by combining alphavirus replicon RNA comprising nucleic acid encoding the pol gene product or immunogenic fragment thereof, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and
b) gradually removing detergent, whereby alphavirus replicon virosomes are produced; and
D) combining the first population of alphavirus replicon virosomes, the second population of alphavirus replicon virosomes and the third population of alphavirus replicon virosomes to produce the population of alphavirus replicon virosomes.
In addition, a method of producing a population of alphavirus replicon virosomes is provided, comprising:
A) (a) producing a first population of alphavirus replicon virosomes by combining alphavirus replicon RNA comprising nucleic acid encoding and env gene product or immunogenic fragment thereof, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and
b) gradually removing detergent, whereby alphavirus replicon virosomes are produced;
B) (a) producing a second population of alphavirus replicon virosomes by combining alphavirus replicon RNA comprising nucleic acid encoding and gag gene product or immunogenic fragment thereof, wherein the gag gene product or immunogenic fragment thereof is modified to inhibit formation of virus-like particles containing the gag gene product or the immunogenic fragment thereof and their release from a cell, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and
b) gradually removing detergent, whereby alphavirus replicon virosomes are produced;
C) (a) producing a third population of alphavirus replicon virosomes by combining alphavirus replicon RNA comprising nucleic acid encoding the pol gene product or immunogenic fragment thereof, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in inactivation of reverse transcriptase activity in the pol gene product or immunogenic fragment thereof, alphavirus glycoproteins E1 and E2, non-cationic lipids and detergent; and
b) gradually removing detergent, whereby alphavirus replicon virosomes are produced; and
D) combining the first population of alphavirus replicon virosomes, the second population of alphavirus replicon virosomes and the third population of alphavirus replicon virosomes to produce the population of alphavirus replicon virosomes of claim 48.
Furthermore, the present invention provides a method of inducing an immune response in a subject, comprising administering to the subject an immunogenic amount of the virosomes of this invention, in a pharmaceutically acceptable carrier.
Also provided is a method of treating or preventing infection by human immunodeficiency virus in a subject, comprising administering to the subject an immunogenic amount of the virosomes of this invention, in a pharmaceutically acceptable carrier.
Additionally provided by this invention is a composition comprising heparin affinity-purified alphavirus replicon particles, wherein the alphavirus replicon particles comprise at least one structural protein which comprises one or more attenuating mutations, as well as a method of preparing heparin affinity-purified alphavirus particles, comprising:
a) producing alphavirus replicon particles, wherein the alphavirus replicon particles comprise at least one structural protein which comprises one or more attenuating mutations;
b) loading the alphavirus replicon particles of step (a) in a heparin affinity chromatography column;
c) eluting the particles from the column of step (b) with a salt gradient (e.g., NaCl gradient); and
d) collecting the fraction from the column which contains the heparin affinity-purified alphavirus replicon particles.
In further embodiments, the present invention provides a method of producing VRP for use in a vaccine comprising:
a) producing a plasmid encoding the nucleotide sequence of an alphavirus replicon RNA;
b) producing a plasmid encoding the nucleotide sequence of one or more helper RNAs;
c) transcribing the plasmids of steps (a) and (b) into RNA in vitro;
d) electroporating the RNA of step (c) into a Vero cell line; and
e) purifying VRP from the Vero cell line of step (d) by heparin affinity chromatography. By this method, VRPs can be produced in large-scale.
In additional embodiments, the present invention provides an isolated nucleic acid encoding a pol gene product or immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof. This nucleic acid can be present in a composition and in a vector. Such a vector can be present in a cell. This nucleic acid can also be present in an alphavirus replicon particle.
The present invention further provides a method of making an alphavirus replicon particle comprising nucleic acid encoding a pol gene product or immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, comprising
a) providing a helper cell for producing an infectious, defective alphavirus particle, comprising in an alphavirus-permissive cell:
(i) an alphavirus replicon RNA, wherein the replicon RNA comprises an alphavirus packaging signal and a nucleic acid encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof, and wherein the replicon RNA lacks sequences encoding alphavirus structural proteins;
(ii) a first helper RNA separate from said replicon RNA, said first helper RNA encoding at least one alphavirus structural protein and furthermore not encoding at least one other alphavirus structural protein; and
(iii) one or more additional helper RNA(s) separate from said replicon RNA and separate from said first helper RNA, said additional helper RNA(s) encoding at least one other alphavirus structural protein not encoded by said first helper RNA;
and with at least one of said helper RNAs lacking an alphavirus packaging signal;
wherein the combined expression of the alphavirus replicon RNA and the helper RNAs produces an assembled alphavirus particle which is able to infect a cell, and is unable to complete viral replication, and further wherein the population contains no detectable replication-competent alphavirus particles as determined by passage on permissive cells in culture;
(b) producing the alphavirus particles in the helper cell; and
(c) collecting the alphavirus particles from the helper cell.
In the method described above, at least one of the replicon RNA, the first helper RNA, and the one or more additional helper RNA(s) can comprise one or more attenuating mutations. The present invention additionally provides alphavirus replicon particle produced according to the above methods.
Further provided is a method of inducing an immune response in a subject, comprising administering to the subject an immunogenic amount of a composition comprising alphavirus replicon particles encoding a pol gene product or an immunogenic fragment thereof of a human immunodeficiency virus, wherein the pol gene product or immunogenic fragment thereof comprises a modification resulting in deletion or inactivation of protease, integrase, RNase H and reverse transcriptase functions in the pol gene product or immunogenic fragment thereof in a pharmaceutically acceptable carrier.